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This file contains supplemental materials and methods describing protein calibrator preparation and pre-analytical variability

This file contains supplemental Figure S2: Temperature stability of analytes

This file contains supplemental Figure S3: Storage stability of analytes

Purpose: To support clinical pharmacodynamic evaluation of the Smac mimetic TL32711 (birinapant) and other apoptosis-targeting drugs, we describe the development, validation, and application of novel immunoassays for 15 cytosolic and membrane-associated proteins indicative of the induction, onset, and commitment to apoptosis in human tumors.Experimental Design: The multiplex immunoassays were constructed on the Luminex platform with apoptosis biomarkers grouped into three panels. Panel 1 contains Bak, Bax, total caspase-3, total lamin-B (intact and 45 kDa fragment), and Smac; panel 2 contains Bad, Bax–Bcl-2 heterodimer, Bcl-xL, Bim, and Mcl1; and panel 3 contains active (cleaved) caspase-3, Bcl-xL–Bak heterodimer, Mcl1–Bak heterodimer, pS99-Bad, and survivin. Antibody specificity was confirmed by immunoprecipitation and Western blot analysis.Results: Two laboratories analytically validated the multiplex immunoassays for application with core-needle biopsy samples processed to control preanalytical variables; the biologic variability for each biomarker was estimated from xenograft measurements. Studies of TL32711 in xenograft models confirmed a dose-dependent increase in activated caspase-3 6 hours after dosing and provided assay fit-for-purpose confirmation. Coincident changes in cytosolic lamin-B and subsequent changes in Bcl-xL provided correlative evidence of caspase-3 activation. The validated assay is suitable for use with clinical specimens; 14 of 15 biomarkers were quantifiable in patient core-needle biopsies.Conclusions: The validated multiplex immunoassays developed for this study provided proof of mechanism data for TL32711 and are suitable for quantifying apoptotic biomarkers in clinical trials. Clin Cancer Res; 22(4); 1000–10. ©2015 AACR.

This file contains supplemental Figure S1: Specificity of antibodies used in multiplex assays

This file contains supplemental Table S2A: Analytical validation- interlaboratory precision, and Table S2B: Fitness-for-purpose clinical utility- intertumoral biological variability

This file contains supplemental Table S3: Fitness-for-purpose clinical utility-Intratumoral biological variability

This file contains supplemental Figure S4: Correlation among multiplexed biomarkers

This file contains supplemental Table S1A: Analytical validation- sample dilution linearity, and Table S1B: Analytical validation- accuracy determined by spike recoveries

Purpose: To support clinical pharmacodynamic evaluation of the Smac mimetic TL32711 (birinapant) and other apoptosis-targeting drugs, we describe the development, validation, and application of novel immunoassays for 15 cytosolic and membrane-associated proteins indicative of the induction, onset, and commitment to apoptosis in human tumors. Experimental Design: The multiplex immunoassays were constructed on the Luminex platform with apoptosis biomarkers grouped into three panels. Panel 1 contains Bak, Bax, total caspase-3, total lamin-B (intact and 45 kDa fragment), and Smac; panel 2 contains Bad, Bax–Bcl-2 heterodimer, Bcl-xL, Bim, and Mcl1; and panel 3 contains active (cleaved) caspase-3, Bcl-xL–Bak heterodimer, Mcl1–Bak heterodimer, pS99-Bad, and survivin. Antibody specificity was confirmed by immunoprecipitation and Western blot analysis. Results: Two laboratories analytically validated the multiplex immunoassays for application with core-needle biopsy samples processed to control preanalytical variables; the biologic variability for each biomarker was estimated from xenograft measurements. Studies of TL32711 in xenograft models confirmed a dose-dependent increase in activated caspase-3 6 hours after dosing and provided assay fit-for-purpose confirmation. Coincident changes in cytosolic lamin-B and subsequent changes in Bcl-xL provided correlative evidence of caspase-3 activation. The validated assay is suitable for use with clinical specimens; 14 of 15 biomarkers were quantifiable in patient core-needle biopsies. Conclusions: The validated multiplex immunoassays developed for this study provided proof of mechanism data for TL32711 and are suitable for quantifying apoptotic biomarkers in clinical trials. Clin Cancer Res; 22(4); 1000–10. ©2015 AACR.

Background: The Division of Cancer Treatment and Diagnostics/National Cancer Institute has embarked on development of a multiplex panel of biomarkers of the apoptosis pathway to support proof-of-mechanism studies of investigational agents targeting apoptosis. The selected biomarkers provide quantitative estimate of the commitment, onset, and induction of apoptosis, especially the intrinsic apoptosis pathway. This study updates progress in development of the multiplex panel. Methods: The immunoassays are built on the Luminex® xMAP multiplex technology platform using magnetic bead capture. The current panel included caspase-3, Lamin-B, Survivin, and markers involved in mitochondrial apoptosis including oligomeric forms of BCL2 family proteins. Calibrators for oligomeric proteins, which are formed in vivo by the non-covalent association of BH3 domain proteins, are developed as synthetic recombinant fusion proteins. The panel is designed for analysis of biomarkers in fractionated cell lysates prepared from flash frozen tumor biopsies. Fit-for-purpose approach is utilized for validation of biomarkers. Results: We have completed development of 15 biomarkers in the panel. Highly specific and sensitive sandwich immunoassays were designed using capture & labeled antibody pairs and recombinant proteins as calibrators. To overcome complications arising from the multiple readout of some markers (due to protein-protein interactions), the multiplex is grouped in three smaller panels: Panel 1(BAK, BAX, Total Caspase-3, Lamin B(intact +45kDa), & SMAC); Panel 2 (BAD, BAX-BCL-2, BCL-xl, BIM& Mcl-1); and Panel 3 (Active Caspase-3, BCL-xl - BAK, MCL-1 - BAK, phospho-BAD, & Survivin). Specificity of oligomeric forms of biomarkers, BAK-MCL1, BAX-Bcl2, BAK-BCL-xl, and BAX-BAK was achieved by capturing BAK and BAX protein and probing with labeled antibodies specific to MCL-1, BCL-xl, BAK, or BAX. Lysates from tumor biopsy samples can be examined in as little as 50 μg of protein. Specificity of the oligomeric assays was also confirmed by immunoprecipitation and immunoblotting techniques. Fit-for-purpose validation studies are ongoing. Conclusions: The development phase of the multiplex panel is completed. The panel is now ready for transfer from the development laboratory to clinical laboratories for first-in-human use. In the next phase, the panel will be launched as commercial assay kits. One unique feature of the panel is the ability to measure novel oligomeric biomarkers that mimic alterations in protein-protein interactions. This dynamic readout of intrinsic pathway proteins could provide much needed direct evidence for activity of important classes of molecularly targeted drugs in early clinical trials. Funded by NCI Contract No HHSN261200800001E. First three authors contributed equally to the study. Citation Format: Apurva K. Srivastava, Soumya Jaganathan, Laurie L. Stephen, Melinda G. Hollingshead, Jason Scull, Robert J. Kinders, Eric Damour, Jennifer Donohue, Adam Layhee, James Mapes, Dominic Esposito, Joseph E. Tomaszewski, Ralph E. Parchment, James H. Doroshow. Progress on development of multiplex panel of 15 biomarkers to support development of anticancer drugs targeting apoptosis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3366. doi:10.1158/1538-7445.AM2013-3366